PROJECT SUMMARY Type 2 diabetes (T2D) is characterized by an abnormal elevation of blood glucose levels resulting from defects in secretion and action of insulin and the incretins. The two incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are secreted from intestinal K and L cells, respectively, in response to nutrient ingestion. Once secreted, GIP and GLP-1 act at pancreatic Beta-cells to enhance glucose-stimulated insulin secretion (GSIS). Although GLP-1-based agents are currently used for the treatment of T2D, significant lapses in our molecular mechanistic understanding of GLP-1 secretion from the intestine and GLP-1 action at the Beta-cell remain. Toward this, the involvement of a new signaling axis involving Cdc42-Pak1 proteins in regulating insulin-induced GLP-1 secretion in vitro was recently revealed. Intriguingly, this pathway resembled the Cdc42-Pak1 pathway our lab discovered in 2007 to regulate second-phase insulin secretion from the pancreatic islet beta cell. However, it remains untested as to whether GLP-1 interfaces with the Cdc42-Pak1 signaling pathway in the Beta-cell to augment insulin secretion. Moreover, insulin is not a canonical GLP-1 secretagogue, such that the physiological relevance of this pathway in the intestinal L cell remains unclear. Thus, the objective of this application is to define the role of the Cdc42-Pak1 signaling pathway in the incretin effect (incretin secretion plus incretin action). I hypothesize that the Cdc42-Pak1 pathway is required for both the secretion of GLP-1 induced by glucose and the enhancement of GSIS by GLP-1, and defects in this signaling pathway contribute to the aberrant glucose homeostasis characterized by T2D. This hypothesis will be tested in three Specific Aims: 1) Establish the requirement of the Cdc42-Pak1 pathway in glucose-induced GLP-1 secretion, 2) Elucidate how GLP-1 interfaces with the Cdc42-Pak1 pathway to potentiate GSIS in Beta-cells, and 3) Delineate the in vivo role of Pak1 signaling in the incretin effect, using a new model of T2D, the Pak1[+/-] heterozygous knockout mouse. Preliminary data show a paucity of Pak1 protein in islets from type 2 diabetic human patients, supporting the physiological relevance of these studies and use of the heterozygous Pak1 knockout mouse. Experimental methods will include siRNA-mediated knockown in intestinal L cells and islet beta cells with rescue strategies, confocal and TIRF microscopy techniques to examine insulin granule mobilization and F-actin remodeling, Rho GTPase activation assays, paired with in vivo and ex vivo analyses of GLP-1 release and incretin-induced insulin secretion. Importantly, my work will provide crucial missing molecular mechanistic data, with future use towards accomplishing my long-term goal of manipulating the incretin system for therapies that more accurately mimic endogenous incretin and insulin secretion to safely restore glucose homeostasis in diabetic patients.